With more than 285 million people currently affected, and an estimated 485 million by 2030, diabetes has become a worldwide pandemic. The healthcare costs associated with this pandemic are staggering with a worldwide cost of $376 billion in 2010 projected to exceed $490 billion by 2030. With 30-40% of diabetics being affected by nephropathy, end stage renal disease (ESRD) is of great concern given the requirement for dialysis or kidney transplantation to sustain the patient's life. There is a pressing need to develop novel therapeutics for preventing or delaying the progression to diabetic ESRD. Activation of non-enzymatic oxidative pathways, forming AGEs (advanced glycation end products) that modify the long-lived collagens of the renal extracellular matrices (ECM), is widely held as a key pathogenic mechanism that underlies diabetic nephropathy (DN). Pyridoxamine (PyridorinTM), an investigational drug candidate, which we have developed over the past two decades, inhibits these pathways under in vitro conditions. Importantly, in two phase 2 clinical trials, Pyridorin (PM) has shown promise in delaying the progression to DN, and it is currently under review by FDA for a phase 3 clinical trial. In the present proposal, we seek to conduct in vivo experimentation to identity detailed pathogenic pathways in the progression of DN and to identify those responsive to PM therapy. Specifically, we will 1) determine changes in the post- translational modifications and molecular composition of renal extracellular matrices (ECM), mesangial matrix and glomerular basement membrane, that accompany or precede development of DN; 2) identify oxidative modifications of renal ECM that confer pathogenic cell-matrix interactions in DN; and 3) explore the urinary metabolome to identify pathogenic pathways and those responsive to therapy. For these in vivo investigations, we will utilize the eNOS-/- C57BLKS db/db mouse model, the most robust DN model to date, which most closely approximates human DN. Furthermore, we will characterize the molecular changes associated with the progression to DN and those responsive to PM therapy using conventional and imaging mass spectrometry and nuclear magnetic resonance spectroscopy, research technologies that can provide precise information about molecular structure of renal tissues. It is anticipated that the findings will yield novel information about pathogenic pathways relevant to DN and PM therapy in humans, and will establish a platform for the development of new drug candidates that are complementary to PM therapy and potentially act synergistically to slow DN progression. PUBLIC HEALTH RELEVANCE: Diabetes will affect an estimated 485 million by 2030, with 30-40% of diabetics developing nephropathy (DN) and end stage renal disease. It is anticipated that the proposed research will yield novel information about pathogenic pathways causing DN and establish a platform for the development of new drug candidates to slow DN progression.